In-vitro culture method and application of crassostrea gigas primary blood lymphocytes

Abstract

The invention discloses an in-vitro culture method of primary blood lymphocytes of crassostrea gigas and application of the primary blood lymphocytes. The in-vitro culture method of the primary blood lymphocytes of the crassostrea gigas comprises the following steps: inoculating suspension of the primary blood lymphocytes of the crassostrea gigas into a culture plate containing a culture medium, and culturing at constant temperature. The culture medium comprises a basic culture medium, glucose, a mixed salt solution, a 1wt% penicillin-streptomycin mixed solution and 50% crassostrea gigas serum, and the pH value of the culture medium is 7.0, and the culture medium is 1000 mOsm. The culture medium for the primary blood lymphocytes of the crassostrea gigas contains 50% of crassostrea gigas serum, simulates the growth environment of the blood lymphocytes, provides nutrient substances, cell factors, antibacterial substances and the like required by the growth of the blood lymphocytes, and is more beneficial to the growth of the primary blood lymphocytes of the crassostrea gigas.

Description

Technical Field

[0001] The present invention belongs to the field of cell biotechnology, and particularly relates to a method for in vitro culture and application of primary hemocytes in the circulatory system of the Pacific oyster (Crassostrea gigas). Background Art

[0002] Shellfish is a general term for mollusks, which is the second largest group in the animal kingdom. They are eucoelomate protostomes with a special evolutionary status and have extremely important scientific value in the study of biological evolution. After a long evolution, shellfish have obtained a unique and efficient innate immune defense system. Hemocytes are the core components of their innate immune system and are immune cells that execute immune defense and immune surveillance. Hemocytes are both the executors of cell-mediated immunity and the main mediators of humoral immunity. Different types of hemocytes play different immune functions during the immune response process and jointly maintain the homeostasis of the body. However, the composition types and functional characteristics of hemocytes in the Pacific oyster are still poorly understood at present.

[0003] The Pacific oyster currently lacks cell lines, which forces the research on its immune defense mechanism to rely on primary hemocytes cultured in vitro. Although the culture of primary hemocytes provides basic materials for research, it has many limitations. For example, the isolated primary hemocytes often contain a large number of microorganisms, which easily lead to medium contamination, and there is insufficient understanding of the specific nutritional factors required by hemocytes, resulting in an extremely unstable in vitro culture system for primary cells and making it difficult to support long-term and systematic functional research. The in vitro culture of hemocytes from marine invertebrates such as shellfish is still a worldwide problem, and it is urgent to optimize its in vitro culture system.

[0004] Primary cells refer to cells that are immediately cultured after being taken from the body. For primary cells cultured in vitro, an appropriate amount of culture medium is added, and they are placed in a suitable culture container and allowed to survive and grow under sterile conditions, appropriate temperature and conditions. Primary cells have a short in vitro time and can well maintain their original basic characteristics. Therefore, establishing a perfect and suitable in vitro culture medium to continue to maintain the characteristics, functional characteristics and vitality of primary cells is a technical problem that needs to be solved urgently by those skilled in the art, providing technical support for hemocyte typing and future research on cell fate determination and pathogen infection. Summary of the Invention

[0005] The present invention provides a culture medium and a culture method for primary hemocytes of the Pacific oyster, aiming to solve the above problems existing in the background art.

[0006] The specific content provided by the present invention is as follows:

[0007] The present invention provides a culture medium for primary hemocytes of the Pacific oyster Crassostrea gigas. The culture medium comprises a basal medium, glucose, a mixed salt solution, a 1 wt% penicillin-streptomycin mixed solution, and 50% Pacific oyster serum, with a pH of 7.0 and 1000 mOsm.

[0008] The 50% Pacific oyster serum means that the volume ratio of the Pacific oyster serum to the basal medium is 1:1.

[0009] The basal medium is selected from Leibovitz L-15 medium suitable for supporting cell growth in a non-CO2 balanced environment.

[0010] The mixed salt solution is composed of a 10% salt solution and a 50% salt solution. The 10% salt solution contains 0.54 g / L KCl, 20.2 g / L NaCl, 0.6 g / L CaCl2, and 3.9 g / L MgCl2, dissolved in deionized water. The 50% salt solution contains 1 g / L MgSO4, dissolved in deionized water.

[0011] In the 1 wt% penicillin-streptomycin mixed solution, the working concentration of sodium penicillin G is 100 U / mL, and the working concentration of streptomycin sulfate is 0.1 mg / mL; it is diluted 100-fold in the culture medium and used after mixing.

[0012] The Pacific oyster serum is obtained by centrifuging and separating the hemolymph of the Pacific oyster. Specifically, the hemolymph of the Pacific oyster is extracted, filtered through a 300-mesh silk screen, centrifuged at 600×g for 5 min at 4°C. The hemolymph is divided into the upper serum and the lower hemocyte mass. The upper serum is taken and filtered through a 0.22-μm filter membrane to remove bacteria, and stored at 4°C.

[0013] Another object of the present invention is to provide the application of the culture medium for primary hemocytes of the Pacific oyster Crassostrea gigas, specifically its application in the in vitro culture of primary hemocytes of the Pacific oyster Crassostrea gigas.

[0014] Another object of the present invention is to provide a method for in vitro culture of primary hemocytes of the Pacific oyster Crassostrea gigas.

[0015] The in vitro culture method is to culture primary hemocytes of the Pacific oyster Crassostrea gigas using the above culture medium.

[0016] The primary hemocytes of the Pacific oyster Crassostrea gigas are cultured using the culture medium. Specifically, 1 mL of the primary hemocyte suspension is inoculated into a culture plate containing 2 mL of the culture medium and cultured at a constant temperature.

[0017] The cell concentration of the primary hemocyte suspension is 10 7 cells / mL. The temperature for constant-temperature culture is 20°C.

[0018] The primary hemocytes in the circulatory system of the Pacific oyster are obtained by centrifuging the filtered hemolymph fluid extracted from the intact blood sinus of the adductor muscle.

[0019] Advantages of the present invention:

[0020] The culture medium for the primary hemocytes of the Pacific oyster provided by the present invention contains 50% Pacific oyster serum, simulating the growth environment of hemocytes, providing nutrients, cytokines, antibacterial substances, etc. required for the growth of hemocytes, and being more beneficial to the growth of primary hemocytes of the Pacific oyster.

[0021] The primary hemocytes cultured with the culture medium provided by the present invention exhibit diverse morphological characteristics. Some cells extend pseudopods and can communicate or migrate rapidly between cells with the help of pseudopods, ensuring stable adherent growth and functional expression of cells in the in vitro culture system, and being more conducive to tracking and observing the differential morphological structures and potential functional characteristics of different types of hemocytes for morphological identification of different types of hemocytes and analysis of their potential functional characteristics. Description of the drawings

[0022] Figure 1 Schematic diagram of obtaining hemocytes from the Pacific oyster sample and the circulatory system used in the present invention and the in vitro culture process.

[0023] Figure 2 Morphological structure diagram of the growth of hemocytes in the circulatory system of the Pacific oyster of the present invention.

[0024] Figure 3 In the examples of the present invention, the culture medium supplemented with serum was used to continuously culture hemocytes for 30 days. Detailed implementation manners

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be fully described below in conjunction with the accompanying drawings in the embodiments of the present application to fully understand the objectives, effects, and application prospects of the present invention. The following examples are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. However, without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps, or culture conditions of the present invention belong to the scope of the present invention.

[0026] The test methods used in the following examples are carried out according to conventional methods or the conditions recommended by the manufacturer without special instructions; the materials, reagents, etc. used are commercially available without special instructions.

[0027] The sources of the reagent consumables used in the following examples are described as follows:

[0028] Leibovitz L-15 medium (ThermoFisher SCIENTIFIC, USA, 21083027); penicillin-streptomycin double antibody mixture (Solarbio LIFE SCIENCES, Beijing, P1400); disposable needle filter (0.22 μm, Pall Corporation, USA, PN 4612); cell culture dish (Corning Corporation, USA, 3506); inverted fluorescence microscope (Carl Zeiss, Germany, Axio Observer A1); laser scanning confocal microscope (Carl Zeiss, Germany, LSM800).

[0029] The primary hemocyte medium of Crassostrea gigas includes Leibovitz L-15 basal medium, glucose, mixed salt solution, serum and antibiotics. The final concentrations of each component are as follows: Leibovitz L-15 basal medium; 50% (v / v) serum; 20.8 g / L glucose; 20.2 g / L sodium chloride; 0.54 g / L potassium chloride; 0.6 g / L calcium chloride; 3.9 g / L magnesium chloride; 1 g / L magnesium sulfate; 100 U / mL sodium penicillin G; 0.1 mg / mL streptomycin sulfate; adjust the pH to 7.0 and the osmotic pressure to 1000 mOsm.

[0030] Example 1 In vitro culture of primary hemocytes in the circulatory system of Crassostrea gigas

[0031] 1. Collection of primary hemocytes in the circulatory system of Crassostrea gigas and preparation of their suspension

[0032] (1) Select 2-year-old Crassostrea gigas, cut the adductor muscle along the inner side of the shell with an oyster knife, and then draw hemolymph from the intact blood sinus with a 20 mL syringe.

[0033] (2) Filter the hemolymph through a 300-mesh silk screen and filter it into a 15 mL centrifuge tube. Centrifuge at 4 °C and 600 × g for 5 min, and collect the upper serum and the bottom hemocyte pellet respectively.

[0034] (3) Add 1 mL of primary hemocyte medium to wash the hemocytes twice, and gently disperse the hemocyte pellet with 1 mL of primary hemocyte medium to obtain a hemocyte suspension.

[0035] (4) Use the primary hemocyte medium to adjust the hemocyte concentration to 10 7 cell / mL.

[0036] 2. In vitro culture of primary hemocytes of Crassostrea gigas

[0037] Inoculate all of the blood lymphocyte suspension obtained in step (4) above into a culture plate containing 2 mL of primary blood lymphocyte medium, and place it in a constant temperature incubator at 20 °C for static culture. Observe under an inverted phase contrast microscope during the culture process.

[0038] In the present invention, the purpose of adding the serum of Crassostrea gigas is to simulate the natural growth environment of blood lymphocytes. The serum is rich in complement components, antimicrobial peptides and other immune molecules, which jointly participate in resisting pathogenic bacteria and can effectively prevent the risk of bacterial contamination of the medium; in addition, the serum contains nutrients necessary for the survival and growth of blood lymphocytes, such as glucose, amino acids, fatty acids, trace elements, ions, hormones, etc., which provide necessary energy for the cells and help maintain the balance state inside and outside the cells. More importantly, the medium supplemented with serum can maintain the characteristics of blood lymphocytes, providing a powerful tool for the typing and functional identification of blood lymphocytes.

[0039] Example 2 Morphological observation and cell classification during the in vitro culture of primary blood lymphocytes of Crassostrea gigas Place the primary blood lymphocytes obtained in point 1 of Example 1 above under an inverted phase contrast microscope (adjust to the DIC module of the microscope) for observation and photography. The results are shown in Figure 2 ... The observation results show that the morphological characteristics of the blood lymphocytes of Crassostrea gigas are significantly heterogeneous. The primary blood lymphocytes with a short in vitro time, resuspended in the in vitro culture medium, can well maintain the original characteristics of different types of blood lymphocytes and can be used for the composition identification and classification of immune cells in the circulatory system of Crassostrea gigas. According to the characteristics of cell shape, the present invention observes that there are mainly cell types such as round / oval blood lymphocytes (according to characteristics such as cell diameter, nuclear-cytoplasmic ratio and granularity, round / oval blood lymphocytes can be further divided into prohemocytes, agranulocytes, semi-granulocytes and granulocytes), dendritic-like blood lymphocytes and amoeboid blood lymphocytes in the blood lymphocytes of Crassostrea gigas. The results are shown in Figure 2 ... The following is a morphological introduction of two special types of cells, dendritic-like blood lymphocytes and amoeboid blood lymphocytes.

[0040] Dendritic-like blood lymphocytes have an irregular shape and extend many dendritic protrusions, greatly increasing the cell surface area, enabling them to effectively capture and process antigens, and being significantly distinguishable from other types of cells based on this unique morphological feature; this type of subtype cell uses its dendritic protrusions to directly contact other types of cells for cell communication (indicated by purple arrows). The results are shown in Figure 2 In higher animals, dendritic cells are identified as professional antigen-presenting cells, which can activate T, B lymphocytes and macrophages, etc., and play an important role in innate immunity and adaptive immunity.

[0041] Amoeboid hemocytes are the only type of cells observed to be capable of rapid migration. These cells have a high degree of deformability and no fixed shape; they change shape through cytoplasmic flow and reorganization of the cytoskeleton. This ability enables them to extend lamellipodia (pseudopods) for movement and perform phagocytic functions, moving by the extension and contraction of pseudopods. Pseudopods are temporary protrusions of the cytoplasm that help cells migrate rapidly on solid surfaces.

[0042] Example 3 Continuous in vitro culture of primary hemocytes of the Pacific oyster

[0043] Continuous culture The primary hemocytes obtained in point 1 of Example 1 above were cultured for 30 days. The cultured hemocytes were significantly superior to the control group without 50% serum addition in terms of morphological characteristics, functional status, and viability. The results are shown in Figure 3 .

[0044] The above-described embodiments merely represent one implementation mode of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A culture medium for primary hemocytes of Crassostrea gigas, characterized in that, It contains a basal medium, glucose, a mixed salt solution, a 1 wt% penicillin-streptomycin mixed solution, and 50% Crassostrea gigas serum, with a pH of 7.0 and 1000 mOsm.

2. The culture medium for primary hemocytes of Crassostrea gigas according to claim 1, characterized in that, The 50% Crassostrea gigas serum has a volume ratio of the Crassostrea gigas serum to the medium of 1:

1.

3. The culture medium for primary hemocytes of Crassostrea gigas according to claim 1, characterized in that, The basal medium is Leibovitz L-15 medium.

4. The culture medium for primary hemocytes of Crassostrea gigas according to claim 1, characterized in that, The mixed salt solution is composed of a 10% salt solution and a 50% salt solution; the 10% salt solution contains 0.54 g / L KCl, 20.2 g / L NaCl, 0.6 g / L CaCl2, and 3.9 g / L MgCl2, dissolved in deionized water; the 50% salt solution contains 1 g / L MgSO4, dissolved in deionized water.

5. The culture medium for primary hemocytes of Crassostrea gigas according to claim 1, characterized in that, For the 1 wt% penicillin-streptomycin mixed solution, the working concentration of sodium penicillin G is 100 U / mL, and the working concentration of streptomycin sulfate is 0.1 mg / mL.

6. The culture medium for primary hemocytes of Crassostrea gigas according to claim 1, characterized in that, The Crassostrea gigas serum is obtained by centrifuging the hemolymph of Crassostrea gigas.

7. The culture medium for primary hemocytes of Crassostrea gigas according to claim 6, characterized in that, For the preparation of the Crassostrea gigas serum, the hemolymph of Crassostrea gigas is extracted, sieved through a 300-mesh silk screen, centrifuged at 4°C and 600×g for 5 min, and the hemolymph is divided into the upper-layer serum and the lower-layer hemocyte mass. The upper-layer serum is taken and filtered through a 0.22-μm filter membrane to remove bacteria, and stored at 4°C.

8. Use of the medium for primary Crassostrea gigas hemocytes according to any one of claims 1-7 in the in vitro culture of primary Crassostrea gigas hemocytes.

9. A method for in vitro culture of primary hemolymph cells of Crassostrea gigas, characterized in that, Take the suspension of primary Crassostrea gigas hemocytes and inoculate it into a culture plate containing the medium according to any one of claims 1-7, and incubate at a constant temperature.

10. The in vitro culture method of primary hemolymphocytes of Crassostrea gigas according to claim 9, characterized in that, The cell concentration of the primary hemocyte suspension is 10 7 cell / mL; the temperature of the constant-temperature culture is 20°C; the primary hemocytes of the Pacific oyster are obtained by centrifugally separating the hemolymph filtered from the hemolymph sinus with intact adductor muscles after extraction.

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